The cerebellum is a large hindbrain structure involved in many aspects of motor control as well as cognitive aspects of behavior. Dysfunction of the cerebellum has been identified to be the primary cause in important motor disorders, notably ataxias, and some forms of dystonia. Nevertheless, at a fundamental level we do not understand how the cerebellum operates. This is in part due to a lack of the appropriate experimental techniques that can manipulate different subunits of the cerebellar network to study its function. Genetic mouse lines are starting to help in isolating specific cerebellar functional involvement in disease, and the present study is using mice to link to this research. The specific innovation of the present proposal consists of using a newly developed optogenetic approach that allows the insertion of channelrhodopsin-2 (ChR2) gene into specific areas or cell types of the brain. ChR2 is a photosensitive ion channel that results in a depolarizing current into neurons expressing it when exposed to blue-wavelength light. We will express ChR2 in the inferior olive of adult mice through adeno-associated viral vector injections, and then study the olivary input to the cerebellum with light stimulation of olivary axons in the cerebellum. This presents an important advance in our ability to dissect cerebellar circuits, as previously electrical stimulation only allowed mixed activation of various fiber pathways. Most importantly, the deep cerebellar nuclei (DCN), which provide the final output from the cerebellum, receive excitatory input from olivary axons, but this functional significance of this pathway has never been isolated. In contrast, Purkinje cells in the cerebellar cortex receive olivary input in the form of climbing fibers, which have been extensively studied as each individual input is extremely strong and can easily be identified as a climbing fiber response. We will determine the effect of olivary input to the DCN using both brain slice recordings and recordings from animals with an intact cerebellar network. The slice recordings will allow us to determine the detailed synaptic properties of the olivary connection to DCN neurons for the first time. The recordings from intact animals will shed light on the impact of this connection in the intact network and whether the same olivary axons exciting a DCN neuron will also lead to a delayed inhibition of the same neuron via Purkinje cell climbing fiber activation. Overall our optogenetic studies address the actions of an important cerebellar input pathway, which hitherto could not be studied with classical techniques. We expect that significant excitatory effects on the DCN balancing inhibitory action of Purkinje cell input will be found. This knowledge will lay the basis for future studies examining the involvement of the olivary input pathway in cerebellar disease states in mouse mutants of ataxia and dystonia. PUBLIC HEALTH RELEVANCE: The cerebellum is the source of debilitating motor disorders, most notably cerebellar ataxias and some forms of dystonia. To better understand and treat these diseases we will study excitatory input to the deep cerebellar nuclei with optogenetic methods allowing a detailed analysis of this pathway for the first time. This analysis will help us to examine changes in cerebellar activity in mouse mutants causing cerebellar disease in future studies. PHS 398/2590 (Rev. 06/09) Page Continuation Format Page